Our long-term goal is to understand the molecular and cellular mechanisms by which the ?2 allele of the apolipoprotein E gene (APOE2) protects against Alzheimer's disease (AD), promotes healthy brain aging, and increases longevity, thereby informing the development of therapeutic strategies for AD and other aging-related conditions. Despite intensive effort to understand why APOE4 is a strong risk factor for AD, the reduced risk associated with APOE2 has garnered much less attention. While APOE2 has been shown to protect against amyloid-? (A?) accumulation, a hallmark of AD pathology, emerging evidence, including our recently published work and preliminary data, demonstrates that APOE2 protects against cognitive decline and increases longevity in the absence of AD pathology. Thus, there is an urgent need to understand how A?-dependent and independent pathways collectively support the function of apoE2 protein in protecting against AD and promoting healthy aging. Given apoE2 in the brain forms larger apolipoprotein particles compared to apoE3 and apoE4 suggesting an overall superior function in transporting lipids, we hypothesize that APOE2 protects against aging-related cognitive decline and AD through hyperlipidation leading to more efficient lipid transport, improved synaptic functions, reduced A? aggregation, and longer lifespan. We will test our hypothesis by employing multidisciplinary approaches including animal and cellular models to address the mechanisms and human studies on a healthy aging cohort to address the relevance. In Aim 1, we plan to analyze how astrocytic apoE2 protects against cognitive decline, synaptic functions and amyloid pathology in an age-dependent manner in our novel cell type-specific and inducible mouse models. We will also breed our experimental animals to the Abca1-KO background thus directly assess how alteration of apoE lipidation impacts the protective effects of apoE2. In Aim 2, we plan to dissect the molecular and cellular mechanisms underlying the protective effects of apoE2 using astrocytes and neurons derived from human induced- pluripotent stem cells (iPSCs). ApoE isoform-specific effects will be addressed both in patient background and under isogenic conditions. Multiple experimental systems including neuron-astrocyte co-cultures and 3-D cultures will be employed for these studies. In Aim 3, we plan to determine the impact of APOE2 on clinical outcomes, biomarker status, and pathological/biochemical measures in humans using a longitudinal Mayo Clinic Study of Aging cohort. Specifically, we will measure and correlate apoE and apoE-associated lipids in cerebrospinal fluid and blood and with clinical, imaging/fluid biomarkers, and neuropathological markers. Together, our proposed studies will uncover molecular and cellular pathways associated with APOE2-mediated protection against cognitive decline and increased longevity, thereby elucidating the mechanisms and informing therapeutic strategies against AD and other aging-related conditions.